Methods and compositions for diseases associated with amyloidosis

ABSTRACT

The present invention generally relates to the detection, treatment or prevention of disease states. Specifically, the present invention relates to the detection, treatment or prevention of amyloidosis or amyloid-associated diseases. The present invention further comprises methods and compositions comprising therapeutic vaccines, antisera and molecular constructs, comprising expression vectors and fusion proteins encoded therein.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/948,049 filed Sep.6, 2001 which claims priority to U.S. Provisional Application No.60/230,391 filed on Sep. 6, 2000 and to U.S. Provisional Application No.60/255,033 filed on Dec. 12, 2000.

FIELD OF THE INVENTION

The present invention generally relates to the detection, treatment orprevention of disease states. Specifically, the present inventionrelates to the detection, treatment or prevention of amyloidosis oramyloid-associated diseases.

BACKGROUND OF THE INVENTION

Amyloidosis includes a variety of diseases characterized by anaccumulation of amyloid material in the organs or tissues of the body.This accumulation can impair vital functions. Diseases associated withamyloidosis include Alzheimer's disease (AD), Down's Syndrome,progressive supranuclear palsy, multiple sclerosis, and Adult OnsetDiabetes. Localized amyloidosis is associated with cognitive decline(senile cerebral amyloidosis; Alzheimer's), heart disease (senilecardiac amyloidosis), endocrine tumors (thyroid cancer), and Adult OnsetDiabetes, diseases which are found in millions of Americans.

A number of impairments specific to amyloid deposits in the brain arelinked with the deposition of the peptide, Aβ peptide (amyloid-βpeptide). Neurological diseases associated with Aβ peptide depositioninclude Alzheimer's, Lewy body dementia, Down's Syndrome, hereditarycerebral hemorrhage with amyloidosis (Dutch type), and the GuamanianParkinsonism-Dementia. Aβ peptide plaques also occur in persons who haveexperienced head trauma and critical coronary disease.

The most common disease related to cognitive decline or dementia isAlzheimer's Disease (AD). This condition is characterized by neuronalloss, neurofibrillary tangles, and neuritic plaques comprised of Aβpeptide. Due to the nature of cerebral amyloidosis, diagnosis ofAlzheimer's before death is difficult and the development of therapiesor other treatments for Alzheimer's have been elusive. Many proposedtherapies are unable to cross the blood-brain barrier in amountsnecessary for effective treatment.

It is currently believed that amyloid plaques in the brain arepredominantly composed of Aβ peptide, a 4 kD protein, 39-43 amino acidslong. Aβ peptide is expressed by a gene located on chromosome 21 and isderived by proteolytic cleavage from the interior of a much larger (770residue) cell protein, amyloid precursor protein (APP). After excision,Aβ peptide is polymerized into amyloid filaments, which in turnaggregate into amyloid plaque deposits. In the brain, these filamentsand aggregates are toxic to neurons and are thought to relate to thecausative symptoms associated with AD.

The inability to examine amyloid deposition of AD in patients beforedeath impedes the ability of researchers to study AD and developeffective therapies targeted at preventing or reversing amyloid plaqueformation on the brain. Damage to CNS neurons due to AD begins yearsbefore clinical symptoms are evident. Prevention of amyloidosis in thebrain would prevent the development of AD. A similar approach topreventing or treating amylin plaque formation in Adult Onset Diabetespatients would also be beneficial.

Other investigations have identified a set of mutations that causefamilial Alzheimer's disease (FAD), in an autosomal dominant manner. Allknown FAD mutations increase either the absolute or relative productionof the most pathogenic Aβ peptide with 42 amino acids. These mutationsarise either in the APP gene itself, affecting the target sites in theAPP where proteolytic cleavage occurs, or in one of two presenilingenes, the protein products which directly or, more probably, indirectlyaffect APP processing. The early step of AD excision is followed by thepolymerisation of the peptide to amyloid filaments, which in turnaggregate into the visible amyloid plaque deposits that characterize thebrains of individuals with AD. These filaments, or possibly intermediateprotofilamants, are toxic to neurons and are thought to lead toneurofibrillary tangles, synapse loss, and neurodegeneration thatunderlie the decline of cognitive functions in Alzheimer's patients.

In addition to the production of the Aβ peptide (amyloid-β peptide),other important steps are involved in the pathogenic pathway leading toAD. Genetic and biochemical studies both assign a key role to a set ofauxiliary proteins that function as “pathological chaperones”. One suchprotein is the anti-protease, anti-chyrmotrypsin, and another is thelipid transport protein, apolipoprotein F, particularly its E4 allelicform. Both of these proteins promote the formation and maturation ofAlzheimer's plaques with their core of Aβ filaments. Specifically, thepathological chaperones can be demonstrated in vitro to accelerate thepolymerisation of Aβ into neurotoxic amyloid filaments, and experimentswith transgenic mice support their role in promoting amyloid formationin vivo. Both proteins are minor components of the amyloid deposits, andare evidently produced as part of an inflammation reaction that arisesin response to the initial diffuse deposition of Aβ peptide.

What is needed are methods and compositions for diagnosis and treatmentof amyloid-associated diseases.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods fordiagnosis, prevention and treatment of disease states associated withamyloidosis. In accordance with the present invention, compositions andmethods are provided that are effective in detecting and treatingamyloid deposition, especially amyloid deposition related toneurodegenerative diseases. In a preferred aspect, amyloid deposition isdetected and treated by compositions comprising a fusion protein,comprised of a segment comprising an antibody or antibody fragment thatbinds to an amyloid peptide epitope and a segment capable of crossingthe blood-brain barrier, such as one or more transferrin moieties.Preferably, the fusion protein composition has amyloid plaque or tangledissolving properties and is capable of crossing the blood-brainbarrier.

Specifically, the present invention provides for the construction anduse of a fusion protein that can detect and treat β-amyloid deposition.A preferred fusion protein is comprised of an anti-beta-amyloidmonoclonal antibody binding region and one or more transferrin moieties.The present invention comprises molecular constructs such as nucleicacid vectors, proteins expressed by such vectors and cells transformedwith such vectors and that express the proteins coded for by suchvectors.

The present invention also comprises methods and compositions comprisinga vaccine that elicits an immune response against amyloid proteins,peptides or fragments, and prevents, stops or hinders amyloiddeposition. The vaccine is comprised of an antigen, an amyloid peptidefragment or epitope that is preferably provided in a liposomal bilayer.In an embodiment of the present invention, an antigen is comprised of amodified amyloid peptide, preferably palmitoylated β-amyloid₁₋₁₆peptide, and methods of administration in a liposomal bilayer.

The invention also provides a method and compositions for preventing oralleviating the symptoms of disease states associated with accumulationor molecular organization of amyloid protein or amyloid plaques ingeneral, including the administration of a pharmaceutically effectiveamount of a derivaterized amyloid peptide or peptide fragment to apatient.

The present invention includes a pharmaceutical composition in an amounteffective to prevent, stop or impede one or more symptoms of a diseasestate involving abnormal accumulation or molecular organization ofamyloid protein, assemblies, fibrils, filaments, tangles, or plaquedeposits. The present invention includes an antigen to stimulateproduction of anti-amyloid monoclonal antibody with the ability todissolve amyloid plaques. The present invention also provides a methodfor treating a disease state associated with amyloidosis, including theadministration of a pharmaceutically effective amount of the vaccine toa patient.

The present invention also comprises methods for making compositionscomprising antisera raised to the epitope of the vaccine compositions ofthe present invention. These antisera compositions are capable ofdissolving amyloid aggregates comprising the epitope or antigen (e.g.β-amyloid). The antisera compositions are effective in the treatment orprevention of the accumulation amyloid protein or the formation ofamyloid assemblies, fibrils, filaments, tangles, or plaque deposits and,therefore, disease states associated with amyloidosis.

The invention further comprises methods and compositions for preventingor alleviating the symptoms of disease states associated withaccumulation or molecular organization of amyloid protein or amyloidplaques including the administration of a pharmaceutically effectiveamount of antisera to a patient.

The present invention is particularly useful in the treatment ofamyloid-associated diseases, preferably, β-amyloid-associated diseases,including Alzheimer's Disease, Lewy body dementia, Down's Syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type), and theGuam Parkinson-Dementia complex or other diseases involving abnormalaccumulation or molecular organization of amyloid protein, assemblies,fibrils, filaments, tangles, or plaque deposits.

This and other objects, features, and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Sequence of Vl chain of 6C6 (SEQ ID NO:4) with sequence sites.

FIG. 2A is a map of vector pET24d+.

FIG. 2B is a linear map of vector pET24d+ (SEQ ID NO:3)

FIG. 3 is a map of vector pET36.

FIG. 4A shows the map of the pBlueBacHIS2 B vector containing the V_(L)fragment.

FIG. 4B shows the map of the pBlueBacHIS2 B vector containing theV_(L)-transferrin fragment.

FIG. 5 is the β-amyloid 1-16 (Aβ₁₋₁₆) peptide anchored to the liposomalbilayer by palmitoylated lysines bound to both ends of the Aβ1-16peptide.

FIG. 6A Synthetic Pathway of dipalmitoyllysine using directpalmitoylation described in Examples 9 and 12.

FIG. 6B Synthetic Pathway of dipalmitoyllysine using activated estersdescribed in Examples 9 and 12.

FIG. 6C Solid Phase Synthetic Pathway of the C-Terminus described inExample 9.

FIGS. 7 A and B are a FAB-mass spectrum of the palmitoylester ofN-hydroxysuccinimide. The expected peak at 623 Da (MH+) and absence ofthe peak for the activated ester (354 Da) are visible.

FIG. 8 is a ¹H NMR spectrum of the palmitoylester ofN-hydroxysuccinimide. The expected signals are visible.

FIG. 9 depicts the disaggregation of β-amyloid fibers by the 6C6=mAB andby sera from mice immunized with palmitoylated Aβ (1-16) reconstitutedin liposomes.

DETAILED DESCRIPTION

The present invention is directed to compositions and methods fordiagnosis, treatment and prevention of diseases associated with amyloiddeposition. Preferred compositions of the present invention includecompositions comprising fusion proteins capable of affecting amyloiddeposits that can cross the blood-brain barrier, and compositions thatare effective in raising an immune response to epitopes of amyloiddeposits. Preferred methods of the present invention comprise use of thecompositions for diagnosis, treatment and prevention of diseasesassociated with amyloid deposition, and for making and using modifiedamyloid peptides and molecular constructs of amyloid genes.

An example of one of the diseases associated with amyloid deposition isAlzheimer's Disease, AD. By definition, AD is diagnosed through theexamination of brain tissue, usually at autopsy. The currentlyrecommended “minimum microscopic criteria” for AD diagnosis is based onthe number of neuritic plaques found in the brain. The amyloid cores ofthese neuritic plaques are composed of β-amyloid protein, also referredto herein as amyloid-β peptide, that is arranged in a predominatelybeta-pleated sheet configuration. Brain amyloid plaques are demonstratedby staining brain sections with thioflavin S or Congo red. Congored-stained amyloid is characterized by a dichroic appearance,exhibiting a yellow-green polarization color. The dichroic binding isthe result of the beta-pleated sheet structure of the amyloid proteins.

It is very difficult to diagnose Alzheimer's disease before death, todevelop drug therapies, or to treat AD. Screening for apolipoprotein Egenotype has been suggested as an aid in the diagnosis of AD.Difficulties arise with this technology, however, because theapolipoprotein E4 allele is only a risk factor for AD, not a diseasemarker. It is absent in many AD patients and present in manynon-demented elderly people. Immunoassay methods have been developed fordetecting the presence of neurochemical markers in AD patients and todetect an AD-related amyloid protein in cerebral spinal fluid. Thesemethods for diagnosing AD have not been proven to detect AD in allpatients, particularly at early stages of the disease. They are alsorelatively invasive, requiring a spinal tap. Recently, radiolabeled Aβpeptide has been used to label diffuse, compact and neuritic typeplaques in sections of AD brain. These peptides, however, do notnormally cross the blood-brain barrier in amounts necessary for imaging.

Congo red may be used for diagnosing amyloidosis in vivo in non-brainparenchymal tissues. But Congo red is probably not suitable for in vivodiagnosis of β-amyloid deposits in brain because only very smallamounts, approximately 0.03% of an injected dose of iodinated Congo red,can enter the brain parenchyma. Radioiodinated bisdiazobenzidinecompounds related to Congo red, such as Benzo Orange R and Direct Blue4, have been proposed to be useful in vitro and in vivo to detect thepresence and location of amyloid deposits in an organ of a patient.However, like Congo red, all of the compounds contain strongly acidicsulfonic acid groups which severely limit entry of these compounds intothe brain.

Attempts have also been made to develop monoclonal antibodies as probesfor imaging of amyloid plaques. For instance, antibodies raised againstthe N-terminal region (1-28) of β-amyloid bind to in vitro-formedβ-amyloid assemblies, leading to disaggregation of fibrils and partialrestoration of β-amyloid solubility. Some of the monoclonal antibodiesraised against soluble β-amyloid (1-28) have also been found to inhibitfibrillar aggregation of β-amyloid peptide in vitro. The success ofthese attempts, however, has been limited due to the difficulty ofgetting these large molecules across the blood-brain barrier. Until thepresent invention, diagnosis, treatment and prevention, of amyloidassociated diseases has had little success.

The present invention comprises methods and compositions for diagnosingand treating diseases and processes mediated by the formation ofabnormal amyloid deposition, such as amyloid plaques. One aspect of theinvention comprises methods of administration of compositions comprisinga fusion protein in a dosage sufficient to detect, and preferablydissolve, amyloid plaques. Another aspect comprising methods of makingand using compositions comprising vaccine or antisera compositions. Thepresent invention is useful in detecting and treating neurodegenerativediseases, such as Alzheimer's Disease. It is also useful in detectingand treating amyloid plaques in Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type) and in theGuam Parkinson-Dementia complex and other amyloid-associated diseases.

An aspect of the invention comprises the construction and administrationof fusion proteins comprised of 1) at least one segment of a bindingregion which binds to an epitope or fragment, preferably an epitope suchas one located in β-amyloid and 2) one or more segments comprisingportions, fragments or whole proteins that are capable of crossing theblood brain barrier, such as one or more transferrin moieties. Thesegment comprising the binding region can be any binding partner that iscapable of attaching, binding or associating with an amyloid-associatedmoiety. For example, the variable region of an antibody, including thelight or heavy chain or both, can be the binding region that is capableof binding an epitope found on an amyloid protein. In a particularaspect of the invention, the variable region of the light-chain of the6C6 anti-β-amyloid mAb was sequenced and cloned with human transferrinto produce a molecular construct that expressed fusion proteins:V_(L)-Transferrin and V_(L)-(Transferrin)₂. The fusion proteins retainedthe property of the antibody 6C6 to solubilize β-amyloid fibers andtangles. The addition of the transferrin moieties allows the fusionprotein to cross the blood-brain barrier.

The present invention also comprises genes which code for these fusionproteins, expression vectors which contain these genes, microorganismsand cells transformed with these expression vectors as well as a processfor the preparation of the genes, proteins, expression vectors andtransformed cells and microorganisms.

The present invention further comprises other methods and compositionsfor preventing or treating diseases and processes associated withamyloidosis. A further aspect of the invention comprises theadministration of compositions comprising a vaccine in a dosagesufficient to elicit an immune response against amyloid epitopes, suchas proteins or peptides, particularly those found in plaques, tangles orother depositions. It also comprises the administration of compositionscomprising antisera raised against amyloid epitopes or immunogenicregions in a dosage sufficient to combat amyloidosis. This invention isparticularly useful for diagnosing, preventing and treatingneurodegenerative diseases associated with amyloid deposition, alsoreferred to as amyloid-associated diseases, such as Alzheimer's, andother conditions associated with localized amyloidosis, such as AdultOnset Diabetes.

The present invention is also directed to methods of inhibitingproduction of Alzheimer-type amyloidosis in a mammal comprisingadministering a pharmaceutically effective amount of a modified amyloidpeptide capable of eliciting an immune response wherein the immuneresponse prevents, hinders, or decreases the amount of amyloiddeposition. The alleviation of symptoms related to amyloidosis followedby administration of derivaterized amyloid peptides, such as thosedescribed herein, results from stimulation of appropriate immuneresponses such that accumulation or formation of the amyloid aggregatesis significantly altered, slowed or prevented.

Amyloid-associated conditions or diseases include, but are not limitedto, Type 2 diabetes mellitus, amyloid A (reactive), secondaryamyloidosis, familial mediterranean fever, familial amyloid nephropathywith urticaria and deafness (Muckle-wells Syndrome), amyloid lambdaL-chain or amyloid kappa L-chain (idiopathic, myeloma ormacroglobulinemia-associated) A beta 2M (chronic hemodialysis), ATTR(familial amyloid polyneuropathy (Portuguese, Japanese, Swedish),familial amyloid cardiomyopathy (Danish), isolated cardiac amyloid,(systemic senile amyloidosises), AIAPP or amylin insulinoma, atrialnaturetic factor (isolated atrial amyloid), procalcitonin (medullarycarcinoma of the thyroid), gelsolin (familial amyloidosis (Finnish)),cystatin C (hereditary cerebral hemorrhage with amyloidosis(Icelandic)), AApo-A-I (familial amyloidotic polyneuropathy-Iowa),AApo-A-II (accelerated senescence in mice), fibrinogen-associatedamyloid; and Asor or Pr P-27 (scrapie, Creutzfeld Jacob disease,Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis)or in cases of persons who are homozygous for the apolipoprotein E4allele.

Fusion Proteins

One aspect of the present invention comprises methods and compositionscomprising molecular constructs and the resulting expressed proteins orcells transformed by such constructs. Compositions comprising molecularconstructs or the expression of such molecular constructs result incompositions of fusion proteins of the present invention comprisingfusion proteins having at least one segment that binds to a portion ofamyloid, such as a peptide, protein, tangle or plaque; and at least onesegment that is capable of crossing the blood-brain barrier. Inparticular, it is preferred that the segment that binds to amyloid is abinding region of an antibody or antibody fragment that binds to anepitope of amyloid, preferably an amyloid peptide or proteins. Moreparticularly, it is preferred that the binding of the fusion protein tothe epitope cause the dissolution of amyloid deposits, preventdeposition of amyloid or detect the presence of amyloid deposits.

Incorporation of genes or other nucleic acid segments, such as codingregions, into expression vectors can be effected using methods known tothe skilled artisan. In this context, reference can be made to thetextbooks of Maniatis et al. (Molecular Cloning, Cold Spring HarborLaboratory, 1982) and Sambrook et al. (Molecular Cloning—A LaboratoryManual, 2nd. ed., Cold Spring Harbor Laboratory, 1989), the contents ofboth being herein incorporated by reference.

Methods for the expression of molecular constructs of fusion proteins inaccordance with the invention are also known to the skilled artisan, andare described in detail in the aforementioned textbooks. The methodsgenerally comprise the following steps:

-   -   a) transformation of a suitable host organism with an expression        vector in which a gene or coding region is operatively bonded to        an expression control sequence;    -   b) cultivation of the host organism under suitable growth        conditions; and    -   c) extraction and isolation of the desired fusion protein from        the host organism. Host organisms include, but are not limited        to, gram-negative and gram-positive bacteria, for example E.        coli and B. subtilis strains, yeast, insect cells, human and        other animal cells.

A preferred method for producing compositions comprising fusion proteinsusing recombinant DNA techniques involves (1) sequencing the bindingregion segment of the fusion protein, for example, the variable regionof a light chain of an antibody, (2) generating a synthetic gene for theexpression of the variable region, (3) ligating the synthetic gene intoa vector, such as the C-terminally His₆-tagged pet24d+ vector or pet36b+vector bearing a C-terminal cellulose binding domain (CBD), (4)expressing this construct in E. coli, (5) fractioning the bacteriallysates, (6) purifying the appropriate fractions using Ni-loadedcolumns, (7) conducting a Western blot of the purified fraction toreveal the desired protein band; (8) amplifying the cDNA of the variableregion of the antibody and the cDNA of human transferrin by PCRtechniques, (9) inserting the cDNA of the variable region and the cDNAof human transferrin into a baculovirus vector, such as pBlueBacHIS2 B,(10) cloning the V_(L)-transferrin and V_(L)-(transferrin)₂ hybrids,(11) verifying the maintenance of the correct coding sequence bysequencing the inserts, (12) analyzing crude extracts of insect cellcultures to verify that they are positive for V_(L)-transferrin bySDS-PAGE, (13) performing Western blot analysis with an anti-His₆ toconfirm these results, (14) purifying the constructs, (15) labeling theconstructs with ¹¹¹In, (15) administering the constructs in anappropriate dosage with a suitable carrier, (16) performing externalgamma-camera and radioactivity counting studies of the brain parenchymaand vasculature to quantify the constructs' capacity to cross theblood-brain barrier and to determine the presence of amyloid plaques.

The fusion protein compositions described herein have therapeuticutility, particularly in treatment of amyloid-associated diseases.Methods of treatment comprise administration of an effective amount of acomposition comprising a fusion protein. The subject is then monitoredfor changes in amyloid plaques or cessation of symptoms. The methods ofadministration comprise one-time administration, sequentialadministrations or long-term bolus or continuous infusionadministrations.

The fusion protein compositions are preferably administered to themammal in a carrier. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed., 1980, MackPublishing Co., edited by Oslo et al. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of apharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the fusion protein,which matrices are in the form of shaped articles, e.g., films,liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of protein being administered.

The fusion protein compositions can be administered to the subject,preferably an animal, mammal or human, by injection (e.g., intravenous,intraperitoneal, subcutaneous, intramuscular), or by other methods suchas infusion that ensure its delivery to the bloodstream in an effectiveform. The compositions may also be administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. Local or intravenous injection ispreferred.

Effective dosages and schedules for administering the fusion proteincompositions may be determined empirically, and making suchdeterminations is within the skill in the art. Those skilled in the artwill understand that the dosage of protein that must be administeredwill vary depending on, for example, the subject which will receive thecomposition, the route of administration, the particular type of fusionprotein in the composition and other drugs being administered to thesubject. A typical daily dosage of the fusion protein in thecompositions of the present invention range from about 1 μg/kg to up to100 mg/kg of body weight or more per day, depending on the factorsmentioned above.

The fusion protein compositions may also be administered to the subjectin combination with effective amounts of one or more other therapeuticagents. It may be administered sequentially or concurrently with the oneor more other therapeutic agents. The amounts of fusion proteincompositions and therapeutic agents depend, for example, on what type oftherapeutic agents are used, the condition being treated, and thescheduling and routes of administration. Following administration ofsuch individual compositions or mixtures of compositions to the subject,the animal's physiological condition can be monitored in various wayswell known to the skilled practitioner.

Fusion protein compositions may further be used in diagnostic assays fordetecting specific antigens or as a nucleic acid probe for, e.g.,detecting the presence or expression in specific cells, tissues, orserum, in vitro or in vivo. Various in vitro diagnostic assay techniquesknown in the art may be used, such as competitive binding assays, director indirect sandwich assays and immunoprecipitation assays conducted ineither heterogeneous or homogeneous phases. See Zola, MonoclonalAntibodies: A Manual of Technicrues, CRC Press, Inc. (1987) pp. 147-158.

The molecular constructs or expressed proteins used in the presentinvention can be labeled with a detectable moiety. The detectable moietypreferably is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed.

In a further embodiment of the invention, there are provided articles ofmanufacture and kits containing compositions comprising fusion proteinsthat can be used, for instance, for therapeutic methods or otherapplications described above. The article of manufacture comprises acontainer with a label. Suitable containers include, for example,plates, slides, bottles, vials, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which includes an active agent that iseffective for therapeutic or non-therapeutic applications, such asdescribed above. The active agent in the composition can comprise themolecular construct or fusion protein. The label on the containerindicates that the composition is used for a specific therapy otherapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

Vaccine Methods and Compositions

Additional methods and compositions of the present invention comprisecompositions comprising at least one modified peptide, fragment orprotein, preferably delivered in liposomal bilayers such as liposomes,that are used in methods to elicit an immune response. Preferably, theseimmune responses are able to overcome immune tolerance to “self”proteins. It is desirable to elicit such immune responses againstamyloid peptides in order to treat or prevent amyloidosis. The productsof the immune response are used to effect amyloid deposits, preferablyin solubilizing amyloid aggregates. The products of the immune response,including but not limited to, antibodies, antisera, stimulated cells andcellular factors, and antigens of modified amyloid nature, peptides,fragments or proteins, comprise compositions of the present invention.

A preferred embodiment of the present invention comprises compositionsof and methods of using a modified peptide, preferably, Aβ₁₋₁₆, toelicit an immune response. The immune response products or the immuneresponse in vivo are used to dissolve or partially dissolve amyloiddeposits. It is desirable to elicit an immune response against β-amyloid1-16 (Aβ₁₋₁₆). A monoclonal antibody, 6C6, developed against the sameepitope, the β-amyloid 1-16 peptide, is capable of solubilizingβ-amyloid fibers and tangles in vitro. This particular embodimentrelates specifically to Alzheimer's disease and conditions associatedwith β-amyloid aggregates, but this invention encompasses derivaterizedamyloid peptides and their use to elicit immune responses capable ofpreventing or impeding conditions associated with amyloidosis in generaland amyloid-associated diseases.

Preferred compositions comprise peptides that are modified by covalentbinding of moieties, such as lipophilic moieties, such moieties arecapable of presenting the peptide on the exterior of the delivery agent,such as anchoring the peptide in the lipid wall of a liposome. Preferredmethods comprise administration into subjects, such as humans, mammalsor other animals, for example, by injection.

Compositions comprising liposomes with modified peptides are used inmethods of vaccination and compositions comprising antisera resultingfrom an immune response to an antigen described herein have therapeuticutility in treatment of amyloid-associated diseases, preferably AD. Thecompositions are preferably administered to the subject having anamyloid-associated disease. It will be apparent to those persons skilledin the art that carriers may be used, depending upon, for instance, theroute of administration and concentration of vaccine or antiseracompositions being administered. The vaccine or antisera compositionscan be administered to the subject by injection (e.g., intravenous,intraperitoneal, subcutaneous, intramuscular), or by other methods suchas infusion to ensure delivery to the bloodstream is in an effectiveform. Additional routes of administration are included to exert local aswell as systemic therapeutic effects.

Effective dosages and administration methods for delivery of thecompositions comprising vaccines or antisera may be determinedempirically and such determinations are within the skill of an artisan.Those skilled in the art will understand that the dosage requireddepends on the subject receiving the protein, the route ofadministration, the particular type of peptide antigen used and othersubstances being administered, among other considerations.

The vaccine or antisera compositions may also be administered to thesubject animal in combination with effective amounts of one or moreother therapeutic agents. They may be administered sequentially orconcurrently with the one or more other therapeutic agents. The amountsof vaccine or antisera compositions and therapeutic agent depend on thetype of therapeutic agents are used, the condition being treated, andthe scheduling and routes of administration, among other considerations.Following administration of vaccine or antisera compositions to thesubject animal, the animal's physiological condition is monitored invarious ways well known to the skilled practitioner.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. Conversely, it is to be clearly understood that resortmay be had to various other embodiments, modifications, and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1 Protein Sequence Analysis of the 6C6 Antibody LightChain (V_(L))

The monoclonal antibody 6C6 (Athena Neurosciences, San Francisco,Calif.) recognizes an epitope located in the 1-16 region of β-amyloidand is capable of solubilizing β-amyloid fibers and tangles in vitro.See Tamaoka et al. 1992, Proc. Natl. Acad. Sci, USA, 89, 1345-9.

Light and heavy chains of 6C6 were separated under reducing conditionsin an agarose gel. An in-gel Lys-C-digestion with Trypsin was performedon the Kappa light chain using 1 mg Lys-C overnight. Fractions wereseparated by reverse phase chromatography (HP-C90-1-IPLC 1 mm×12.5 cm,DAD-detector). The mass of collected fractions was determined by massspectrometry (MALDI-TOF). N-terminal sequencing was performed using aPE-Biosystems-sequencer (Procise ABI 492, Langen, Germany). Otherfragments were obtained by in-gel digests with the endoprotease V-9cleaving C-terminal of Glu. Peptides were sequenced starting at theN-terminus. The sequence of the V_(L) chain is given in FIG. 1 and SEQID NO:4.

Example 2 Gene Synthesis and Cloning of the V Region of the Light Chain(V_(L))

The amino acid sequence of the fragment V_(L) was converted into anucleotide SEQ ID NO: 1 and inserted into an appropriate vector system.SEQ ID NO:1 includes a methionine residue at position 0 as well as theleucine and glutamine residues at position 109 and 110 as a result ofthe cloning strategy. The actual DNA sequence of the V_(L) chaincorresponds to SEQ ID NO:4. The V_(L) gene fragment was synthesizedusing a PCR-based approach. The sequences of the sense and anti-sensestrands were divided approximately into 25 nt oligonucleotides at the5′-end (external oligo) followed by four 50mers (internal oligo) in agapless manner. Internal oligonucleotides were mixed and used astemplate in a PCR with the two external 5′-oligos as primers, in a100-fold excess. PCR was performed in 500 volume using 1 unit of aproof-reading polymerase (Ultma, Perkin-Elmer) with 2 mM MgCl₂, buffersas supplied by the manufacturer and 25 pmol of each external primer. Thereaction started 2 minutes after incubation at 99° C. in a Sanyothermocycler by addition of the polymerase going through 30 cycles at95° C. for 2 minutes, 66° C. for 2 minutes, and 72° C. for 1 minute. TheV_(L) gene sequence was verified by gel electrophoresis.

The PCR product was cleaved by NeoI and XhoI. The insert was prepared byagarose gel electrophoresis and ligated into a prepared pET24d+ vectoror pET36b+ vector (Novagen), respectively. Initially the plasmid pET24d+was used to insert the gene fragment (V_(L)) next to a hexa-histidinetag in order to detect and purify the fragment in subsequentexperiments. Unfortunately, the anti-His-tag antibodies did not actsufficiently in the detection procedure showing high non-specificbinding in Western blot analysis. Therefore, a second expression vector,pET36b+ containing a cellulose-binding domain (CBD) of a prokaryoticcellulase and several short peptide tags was used in order to purify theprotein. The CBD included a propeptide acting as signal for thetranslocation of the CBD to bacterial periplasm. The plasmid pET24d+containing V_(L) was named pETVL102; the plasmid pET36b+ with the V_(L)insert was named pETVL112. The maps of both vectors are given in FIGS. 2and 3.

Example 3 Expression in E. Coli

E Coli B21 (DE3)pLysS were transformed with the plasmid pETVL102(pETVL112) and the empty vector pET24d+ (pET36b+) as a control.Overnight cultures were used to inoculate 50 ml of LB-medium containing50 μg/ml kanamycin and 30 μg/ml chloramphenical. Cultures were grown at37° C. for 2 hours and agitated at 200 rpm. Expression was induced bythe addition of IPTG (final concentration 0.5 mM). 2 ml samples weretaken after 2 hours and 4 hours and centrifuged (5 min, 1600×g, 4° C.).The pellets were washed with 1 ml of TrisHCI buffer (25 mlM, pH=7.5) anddissolved in 200 μl of 1×SDS gel-loading buffer (RotiLoad 1, Carl Roth).The samples were sonicated, heated (5 minutes, 95° C.) and analyzed on a15% SDS-PAGE.

In a subsequent step, expression of the V_(L)-fragment in pETVL112 wasrepeated on a 2.5 L scale. The total soluble cell fractions weresubjected to immobilized nickel affinity chromatography (Ni-1MAC) in aFPLC system. A column of Chelating Sepharose FF (Pharmacia Biotech)charged with Ni2+ ions was equilibrated with Buffer A (25 mM TrisHCl,0.1 mM NaCl pH=7.5).

A 3 ml sample was loaded, followed by washing with Buffer A containing20 mM imidazol•Hel. Elution was accomplished with Buffer A containing500 mM imidazol•HCl and the fractions were analyzed in a Western blot.

The rest of the soluble cell extract of E. Coli pETVL112 was treated asdescribed above and fractions around n^(o) 58 were pooled. This pool wassubjected to a second affinity chromatography using a cellulose matrix.The CBD binds to cellulose in the presence of salt and is eluted eitherby pure water or by an organic solvent, e.g. ethylene glycol. The pool(20 ml) was forced through two columns (CBinD. 900 Cartridges, Novagen).Then, the columns were washed with 10 ml wash buffer (20 mM TrisHCl,0.8M NaCl, pH=7.5) and 2 ml loading buffer (20 mM TrisHCl, pH=7.5). Eachcolumn was eluted with 1 ml 100% ethylene glycol. The flow-through, thewashing solution and the eluate, was analyzed in Coomassie blue-stainedSDS-PAGE and Western blot.

In sonicated cell fractions only the soluble fraction showed thespecific 30 kDa signal. It was concluded that the V_(L)-fragment mightbe very unstable even if fused to a stable moiety, and that it was notdeposited as intracellular inclusion bodies, but processed correctly(unprocessed fusion protein has a calculated mass of about 34 kDa) andtranslocated to the host's periplasm. For the verification of theseresults the expression of V_(L) in pETVL112 was repeated on a largervolume. The results were collected by affinity chromatography on a FPLCsystem. The absorption was monitored at 290 nm. The imidazol stepgradient was used. The specific elution peak was seen at approximately120 ml total elution volume.

Eluate fractions were analyzed in a Western blot. 7.5 ml of fraction 8,fraction 25, fraction 33, and fraction 58 of the pETVL112 strain and thecontrol (even-numbered lanes) were separated on a 12% SDS-PAGE,transferred to nitrocellulose, incubated with anti-CDB-antiserum, andassayed by protein A-peroxidase conjugate and chemoluminescence. Thespecific signal was seen in lane 7. Other signals are ambiguous due to asecond antiserum that was used to detect the molecular weight marker(M).

The Coomassie blue-stained SDS-PAGE showed no band at the expectedposition. This suggests a very small amount of the fusion protein ispresent after this first chromatographic step relative to the total.

The rest of the soluble cell extract of E. Coli pETVL112 was treatedsimilarly and a collection of fractions was forced through columns.Eluate fractions were analyzed by Western blot. The resulting eluate wasalmost without cross-reacting signals. On the Coomassie blue-stained gela very faint signal at 30 kDa was visible. See SEQ ID NO:3 and FIGS. 2Aand 2B.

Example 4 Construction of V_(L)-Human Transferrin Hybrids

Various V_(L)-transferrin hybrids were ligated and cloned into thebaculovirus vector pBlueBacHIS2 B (4.9 kb, Invitrogen) for expression ininsect cells. The V_(L) fragment. (pETVL102) and cDNA for humantransferrin (pUC18/hTF, human full length transferrin, 2.3 kb in pUC18vector, 2.7 kb, obtained from R. T. A. MacGillivray, Department ofBiochemistry and Molecular Biology, Vancouver, Canada) were amplified byPCR. The introduction of stop codons and restriction sites at the end ofthe fragments were done using modified primers.

The following fragments containing either the cDNA of V_(L) or the cDNAof human transferrin, stop codons, and appropriate restriction siteswere successfully amplified:

-   -   BamH I-V_(L)-XhoI    -   Xho Ihtf-STOP-Sal I    -   Xho I-hTF-Bgl II    -   Bgl II-hTF-STOP-Sal I        Cleavage of the amplified DNA fragments by restriction enzymes        allowed the ligation of the purified V_(L) and transferrin        fragments in the following manner:

1. BamH I-V_(L)-Xho I x Xho I-hTF-STOP-Sal 1

2. BamH I-V_(L)-Xho x Xho I-hTF-Bgl II x Bgl II-hTF-STOP-Sal 1

Cleavage 1 yielded a fragment containing V_(L)-TF which was ligated bythe restriction site Xho 1 and ended by a stop codon.

In cleavage 2, V_(L) is connected with transferrin over Xho I followedby a second transferrin connected to the first over the restriction siteBgl II. The fragment ends with a stop codon.

The V-region of the light chain with one transferrin moiety (V_(L)-TF)as well as the V-region with two transferrin molecules (V_(L)-(TF)₂)were cloned into the multiple cloning site of the baculovirus vectorpBlueBacHIS2 using the restriction sited BamH 1 and Sal I. Insertion ofthese two selected recombinant clones were verified before sequenceanalysis.

Starting from pETVL102 the V_(L) was amplified by PCR using aDNA-polymerase with 3′->5′ proofreading-activity. By usingV_(L)-specific primers with 5′-terminal extensions, recognition sitesfor restriction enzymes (5′-end: BamH I, 3′-end: Sal 1) and a STOP-codonfollowing the last amino acid of the V_(L)-fragment were added to thePCR-product.

The purified V_(L)-PCR product and the vector pBlueBacHIS2 B werecleaved with the restriction enzymes BamH I and Sal 1. The restrictedvector was dephosphorylated using calf intestine phosphatase (CIP). Therestriction products were identified by agarose gel electrophoresisbefore the V_(L) fragment was ligated with the CIP-treated pBlueBacHIS2B vector. The T4 DNA-ligation and CIP treatment were performed asdescribed by the manufacturer (MBI Fermentas). See FIGS. 4( a) and (b).

Example 5 Transformation, Screening and DNA-Sequencing of the SubclonedV_(L) with the Baculovirus Vector pBlueBacHIS2 B

Competent E. Coli XL-1 Blue MRF cells (Stratagene) were transformed bythe ligation product pBlueBacHIS2 B containing the V_(L)-fragment byapplying routine procedures (Sambrook et al., 1989. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory, New York). After thetransformation, six of the resulting clones were tested for the presenceof the V_(L) insert.

The analysis of the single colonies was done by a PCR-based techniqueusing the same V_(L)-specific primers, which were previously used forV_(L)-amplification. Verification of the construct was done byrestriction cleavage and sequencing of the inserts.

The BamH I/Sal I-restricted V_(L)-fragment was ligated with BamH I/SalI, digested, and CIP-treated pBlueBac HIS2 B vector.

After transformation, six of the resulting clones were tested for thepresence of the V_(L)-insert. Analysis of single colonies shows that allsix clones contained the V_(L)-insert. Clone “V_(L)-1” was selected forfurther analysis by DNA-sequencing.

The maintenance of the correct coding sequence was verified bysequencing the inserts pBlueBacHIS B-V_(L), pBlueBacHIS B-V_(L)-TF andpBlueBacHIS B-V_(L)-TF₂. The maps of the pBlueBacHIS2 B containing theV_(L)-fragment and V_(L)-transferrin are shown in FIGS. 4 (a) and (b).

Example 6 Generation of the Recombinant Baculovirus and Expression ofthe Proteins V_(L), V_(L)-TF and V_(L)-(TF)₂

Expression of the fused proteins V_(L)-TF and V_(L)-(TF)₂ as well asV_(L) was performed in E. coli. The proteins were analyzed in aCoomassie-stained gel. Three selected plasmids, pBlueBacHIS2 BVL-2,pBlueBacHIS2 BVL-Tf-4 and pBlueBacIIIS2 B V_(L)-TF2-5, were verified byDNA sequencing before the in vivo recombination of transfer vector andvirus DNA was performed (Ba N-Blue™ Linear DNA, Invitrogen).

Sf9-cells were transfected by the plasmids pBlueBacHIS2 BVL-1,pBlueBacHIS2 BVL-TF-4 and pBlueBacHIS2 B. V_(L)-TF2-5 as well as byacceptor-DNA (Bac-N-Blue TM-DNA, Invitrogen). The supernatants werecollected after 3 and 5 days and tested in dilutions of 1:10 to 1:10,000on agarose/X-gal-overlay plates. After 5 days of incubation, viruseswere isolated from 8 plaques.

Sf9-cells were infected with these viruses (four samples per plaque) andsupernatants were collected 4 days later (named P1-lysates) in order toisolate viral DNA. The DNA was characterized by PCR-technique.

Example 7 Analysis of the Fused Proteins Expressed by the BaculovirusVector System

For the generation of lysates with a high titer of viruses, 3×10⁷Sf9-cells were infected with the PI lysates of V_(L), V_(L)-TF andV_(L)-TF₂ with a m.o.i (multiplicity of infection) of approximately 0.1.After 3 days the supernatants were collected and their titers determinedby limited dilutions. The proteins were isolated and characterized in aCoomassie-stained 10% PAGE-gel.

The PCR with V_(L) showed the expected fragments as well as V_(L)-TF ina Coomassie-stained gel. The amplification products of V_(L)-TF₂ (lightchain with 2 transferrin moieties) seem to have lost one of thetransferrin residues showing a length of the product of V_(L)-TF or hadlost an even longer fragment. Because of the instability of V_(L)-TF₂,the virus DNA of four other clones was analyzed by PCR. Unfortunately,no V_(L)-TF₂ with the correct length was detected.

A virus titer of 2×10⁸ pfu/ml (plaque forming units) for V_(L)-1 (26ml), 1×10⁸ pfu/ml for V_(L)-TF4 (26 ml), and 1×10⁸ for V_(L)-TF₂ (14 ml)was obtained from the three selected baculovirus clones V_(L), V_(L)-TF,and V_(L)-TF₂. From the V_(L)- and V_(L)-IF-infected cells, proteinswith the expected molecular weights of 15 kD and 80 kD could beextracted. The extract of V_(L)-TF₂ showed two proteins with themolecular weight of 80 kD and about 105 kD, both not the relevantprotein of V_(L) fused to two transferrin molecules. Western blotanalysis of crude extract of insect cells cultures containing V_(L) andV_(L)-transferrin performed with an anti-His₆ confirmed these results.

Example 8 In Vivo Administration

High grade purification (>95%) of the constructs is performed. Asufficient amount of V_(L) and V_(L)TF is obtained, and the constructsare labeled with ¹¹¹In. External γ-camera and radioactivity countingstudies of the brain parenchyma and vasculature allows the quantitationof the constructs' capacity to cross the blood-brain barrier.

Example 9 Preparation of Palmitoylated Lysines for Solid Phase PeptideSynthesis

The following methods were used to produce vaccine compositions.Palmitoylated lysines were appended to both ends of the Aβ 1-16 peptideto anchor the β-amyloid 1-16 (Aβ₁₋₁₆) peptide in the liposome bilayer.See FIG. 5.

Two strategies were adopted and two tetrapalmitoylpeptides weresynthesized by means of an Applied Biosystems peptide synthesizer: 1)the N-terminus has two palmitoyl residues, (i.e. α,ε-dipalmitoyllysine)while at the C-terminus two a-palmitoyllysines were insertedsequentially; 2) four a-palmitoyllysines are inserted, two at each end.For the automated solid phase synthesis of the peptide the first aminoacid, a-palmitoyllysine, was N-protected and anchored on the4-alkoxybenzyl alcohol resin by means of an ester linkage.Fluorenylmethoxy-carbonyl (Fmoc) group was preferred as the a-amineprotecting group for lysine and its derivatives. Methods for making suchbuilding blocks are known. See FIGS. 6 a and b.

Example 10 Anchoring of FmocLys(PAL) on the Alkoxybenzyl Alcohol Resin

FmocLys(Pal)OH (from BACHEM) was reacted with the alkoxybenzyl alcoholresin (from BACHEM) in the presence of dicyclohexylcarbodiimide (DCC,Aldrich) and dimethyl-aminopyridine (DMAP, Aldrich) in dry, freshlydistilled methylene chloride according to the procedure optimized by G.Lu et al., J. Org. Chem., 46, 3433 (1981). After stirring for threehours at room temperature, the reaction mixture was filtered and washedthoroughly ten times with dry methylene chloride. To ensure completereaction, the obtained resin was reacted once more with a fresh portionof FmocLys(Pal)OH, in the presence of DCC and DMAP in dry methylenechloride at room temperature overnight. The next day, after filtrationand washing with methylene chloride the resin was dried in vacuum. TheFT-IR spectrum of the product shows the expected bands for the esterlinkage at 1720 cm⁻¹, for the NH group at 3327 cm⁻¹ and for the amidecarbonyl group 1626 cm⁻¹. All these bands are absent in the FT-IRspectrum of the starting alkoxybenzyl alcohol resin. The final quantitywas 227 mg with a loading of about 0.5 mmol/g. See FIG. 6 (c)

Example 11 Solid Phase Synthesis

To the resin, the second palmitoylated lysine was added by means ofFmocLys(Pal)OH after deprotection (removal of the Fmoc group). Then 16cycles of synthesis were performed for the β-amyloid. For test purposes,a small quantity of this peptide was then cleaved from the resin andinvestigated by electrospray mass spectrometry (ES-MS). The crudemixture shows the presence of three peptides, the major component beingthe desired one having peaks at 896.9, 673.0 and 538.0 Da correspondingto the +3, +4 and +5, respectively charged molecular ions. The molecularion is absent in the spectrum, but can be inferred from this series tobe at 2687.86 Da. The other two components correspond to peptides havingone or two palmitoylated lysines less (367 Da mass difference) withmolecular ions inferred at 2321.43 and 1953.87 Da. This indicates thatthe coupling of the FmocLys(Pal)OH was incomplete.

α,ε-dipalmitoyllysine was coupled to the rest of the uncleaved resin.Even prolonged coupling times (overnight at room temperature) leftunreacted material (ninhydrin test) due to the low solubility in DMF ofthe dipalmitoylated lysine. After cleavage from the resin, the ES-MSshows that the desired tetrapalmitoylpeptide (M+=3292.43) is presentonly in about 20%.

In a second run, to avoid the sluggish coupling withα,ε-dipalmitoyllysine, after the 16 cycles which apprehended theB-amyloid residues to the first two palmitoyllysines were completed, twosequential palmitoyllysines were inserted at the end, the coupling beingperformed twice for each. The ES-MS spectrum after cleavage from theresin (with TFA) shows the desired peaks of the tetrapalmitoylpeptide asthe M4+ at 855.4 Da and M5+ at 05.4 corresponding to a molecular ion of3421.65. This peptide amounts to about 35% or the mixture, the maincomponent being a peptide having two Lys(Pal)-residues less (M+ at2688.58 Da). A minor component has an extra lysine missing (M+ at2560.42 Da).

The mixture or peptides was composed of the desired tetrapalmitoylatedpeptide and peptides lacking palmitoyl residues, thus making the doubleinsertion into liposomes improbable. Therefore, we decided to use themixtures for liposome preparation.

Example 12 Preparation of α,ε-dipalmitoyllysine

Direct palmitoylation or lysine with palmitoyl chloride in aSchotten-Baumann reaction with aqueous sodium hydroxide, on a 20 g scaleled to a material which contained appreciable amounts of palmitic acidand which could not be separated from the desired a,e dipalmitoyllysine.An indirect method had to be adopted following the procedures of H.Kiwada, et al., Chem. Pharm. Bull., 35, 2935-39 (1987); Y. Lapidat, etal., J. Lipid Res., 9, 142-44 (1967).

The palmitoylester of N-hydroxysuccinimide was synthesized first frompalmitic acid (Fluka), N-hydroxysuccinimide (Aldrich) in the presence ofDCC (Aldrich) in ethyl acetate in 77% yield. This activated ester wassubsequently reacted with the sodium salt of lysine in aqueoustetrahydrofurane. The crude product obtained after filtration andwashing with water still contained some unreacted activated ester as putinto evidence by proton NMR and FAB (Fast Atom Bombardment)-massspectra. Recrystallization from chloroform provided a pure sample of 640mg. Its FAB-mass spectrum shows the expected peak at 623 Da (MH+) andabsence of the peak for the activated ester (354 Da). See FIG. 7 andFIG. 8.

Example 13 Reconstitution of the Palmitoylated Peptides in Liposomes

Liposomes with Lipid A were used as adjuvants in an attempt to break themouse immune tolerance to Aβ 1-16. They were prepared by mixingdimyristoyl-phosphatidylcholine, dimyristoylphosphatidyl-glycerol, andcholesterol (Avanti Polar Lipids, Alabaster, AI, USA) in the molarratios 0.9, 0.1:0.7. Monophosphoryl lipid A, a strong immunomodulator,(IASL Biologicals, Campbell, Calif., USA) was added at a concentrationof 40 mg per mmole of phospholipids. Tosi et al., Biochem. Mophys. Res,Com., 212, 494-500 (1995). The palmitoylated peptides were added at amolar ratio to phospholipids of 1:100 and 1:200. Solvents wereevaporated. The resultant film, after hydration with sterile phosphatebuffer saline (PBS, pH 7.4) with a final phospholipid concentration 4mM, was further homogenized by orbital shaking. The liposome suspensionwas mixed with sterile Alum 15 minutes before injection (9:1 vol:vol,Rehydrogel, HYA, Rebeis Inc, Berkley Heights, N.J.).

Example 14 Immunization of Mice

Eight BALB/c mice (Charles River Laboratories, Wilmington, Mass., USA)were immunized by 6 i.p. innoculations at two week intervals with 200 μLof the palmitoylated peptide-liposome/Alum suspension. One group of 3mice was immunized according to the same protocol with PBS/Alum only.Another group of 3 mice were immunized according to this protocol, with4 mM phospholipids in PBS and Alum, without palmitoylated Aβ 1-16. Bloodwas collected from the tail vein 4 days after injection. The collectedblood (10-30 μl) was diluted immediately with 10 μl PBS and 5 μlheparin. The samples were centrifuged, the serum was removed and testedin an ELISA assay.

A second group of 19 transgenic mice (NORBA, Hoechst Marion Roussel,Bridgewater, N.J.) of different ages, constitutively present β-amyloidplaques on their pancreas. They were immunized using the same protocoldescribed above. Before sacrificing, the immunized NORBA mice received a5^(th) immunization. Blood was collected and assayed for anti->amyloidantibodies in an ELISA assay.

Further, 12 B57131/6-mice were immunized as described before. In thisgroup liposomes and palmitoylated β-amyloid (1-16), liposomes; mixedwith scrambled β-amyloid (42-1) and liposomes were used for injection.Alum was added as before. As an additional control 3 mice were immunizedwith liposomes and palmitoylated β-amyloid (1-16), but without Alum.Blood was collected and tested as described.

Example 15 ELISA Enzyme-Linked Immunosorbent Assay) Experiments

Microtiter plates were coated with 50 μl of β-amyloid 1-28 solution (1mg/ml) overnight at 40° C. Blocking of the wells by 200 μl BSA/PBS (0.5%BSA) for two hours at 37° C. followed before washing with 200 μl ofPBS/0.005% Tween 20.

Various dilutions of serum (1:100-1:100,000) were incubated for 2 hoursat 370 C. The plates were then washed twice with 200 μL of PBS/0.005%Tween 20 before 50 μl of a goat-anti-mouse-antibody (alkalinephosphatase conjugated) in a 1:30,000 dilution was added. After 2 hoursat 37° C. the wells were washed as described above. Then, 100 μl of thesubstrate (PNPP, paranitrophenyl phosphate, 1 tablet in 5 ml distilledwater) was added and absorption was measured at 405 nm by an ELISAreader 30-60 min later.

After the third injection with liposomes/palmitoylated β-amyloid (1-16),ELISA assays showed a significant immune response in the vaccinatedBalb/c mice. The antibodies were specific to the injected antigen. TheOD₄₀₅ of 1:5000-dilutions of the collected sera were 10 to 20 foldhigher than those of controls which were dilutions of untreated mice.Thus, this immunization procedure elicited an immune response against Aβ1-16 in mice. No immune response was detected in the mice havingreceived control immunizations.

The same immunization protocol was used for inoculation of NORBAtransgenic mice to study binding to and possibly dissolving ofβ-amyloid-plaques on their pancreases in vivo. 19 transgenic mice(NORBA, Hoechst Marion Roussel, Bridgewater, N.J.) of different ages,constitutively present β-amyloid plaques on their pancreas, wereimmunized. Before sacrificing, the immunized NORBA mice received aseventh immunization. Blood was collected and assayed for anti-β-amyloidantibodies in an ELISA assay. In 1:5,000 dilutions of the sera the OD₄₀₅was 10 fold higher than in controls. The control NORBA mice did not showany anti-Aβ antibodies in their sera. The 3rd group of immunized C57Bl/6mice receiving Aβ 1-16 as well as the scrambled Aβ 42-1 and as acontrol, liposomes without a peptide reached a tenfold higher titer in a1:10,000-dilution of the sera versus control. See Tables 1 and 2.

TABLE 1 ELISA of sera of Immunized Ba1b/c and C57B1/6 Mice Palm Palmβ-Amyloid Scrambled β-Amyloid (1-16)/ β-Amyloid (L-16)/ liposomes,(42-1)/ Lipo- liposomes, Untreated lipid A liposomes, somes, lipid Aanimal Antigen in C57B1/6 lipid A lipid A in Ba1b/c Ba1b/c 10000* 0.160.06 0.011 — — 5000 0.22 0.01 0.012 0.22 0.01 2500 0.46 0.024 0.060 0.300.04 1000 0.61 0.011 0.041 0.31 0.03  500 0.99 0.044 0.039 0.46 0.04 100 — — — 0.60 0.03 OD₄₀₅ Dilution: 1-* Secondary antibody: alkalinephosphatase conjugated goat anti mouse antibody. Substrate (PNPP)-p-nitrophenyl phosphate. Assay was performed after the 3. (C57BV6) andafter the 4. (Ba1b/c) booster injection. For each antigen/adjuvants 3animals were injected.

TABLE 2 Immune response in mice inoculated with palmitoylated β-amyloid(1-16), scrambled β-amyloid (42-1) in combination with liposomes/lipid Aand Alum and liposomes without amyloid. Phospho- Antigen palm.-A_(β)1-16A_(β) 42-1 lipids number of C57B1/6 mice with 2/3 0/3 0/3 a pos. titerin 1:10000 after 5 Injections dilution of sera number of Ba1b/c micewith a 3/3 0/3 positive titer in 1:5000 after 4 injections dilution ofsera number of transgenic 7/9 NORBA mice with a pos. (Aventis) titer in1:5000 dilution of sera after 7 injections *OD 405 was tenfold of thecontrol (only phospholipids)

Example 16 Pathology Investigation of the Immunized C57Bl/6 Mice

The immunized C57Bl/6 mice underwent a pathological investigation afterthe immunization procedure was finished. Sections of kidney, liver,heart, bone marrow, pancreas, spleen and brain were analyzed.

Example 17 Immunohistochemical Investigation of the Pancreas ofVaccinated NORBA Mice

23 NORBA mice of different ages containing β-amyloid-positive andβ-amyloid-negative animals were immunized with palmitoylated Aβ (1-16)reconstituted in liposomes, as described (19 mice) or with liposomesonly (4 mice). These animals have the β-amyloid plaques on the pancreasinstead of brain. The vaccinated mice were sacrificed after the 7^(th)injection and their pancreases were collected and preserved in formalin.The preserved pancreas pieces were soaked in sucrose solution to preparethem for frozen sections. The thin sections obtained were analyzed byThioflavin T, a fluorescence dye specific for B-amyloid aggregatesstaining (Vassar and Culling. 1959), to detect B-amyloid on the surfaceof the pancreas. The sections were analyzed also using a FITC-labeledanti-β-amyloid antibody (Accurate Chemical Co., Westbury. Conn.). Thebinding of the mouse “autoantibodies” to the β-amyloid plaques wasassayed with a rabbit-antimouse IgG, FITC-labeled.

A histological study of Thioflavin T stained sections of pancreases fromNORBA transgenic mice, vaccinated with palmitoylated Aβ (1-16)reconstituted in liposomes was performed. The stained pancreatic tissueof a B-amyloid negative animal showed a diffuse, weak, backgroundfluorescence with some bright spots which represented vessels. Theacinar cells were completely dark. An 18-month old-animal with fullydeveloped B-amyloid plaques showed intense fluorescence throughout theacinar cell fields. There was also very bright fluorescent areassuggesting larger blood vessels. A 18-month old mouse with fullydeveloped B-amyloid plaques, which was vaccinated and examined 4 monthsafter first inoculation, showed focal (patchy) areas of fluorescenceamong acinar cells, but predominantly many patches of non-fluorescentacinar cells. The results showed that the vaccination eitherdisintegrated β-amyloid plaques or reversed their deposition.

Quantitative evaluation of the average fluorescence intensity in eachsection stained with Thioflavin using a luminosity analysis software,indicated that the pancreas sections from 19 months old NORBA vaccinatedanimals showed less than 25% of the high intensity fluorescence of thesame animals, unvaccinated. A sampling of these quantitations is shownin Table 3.

TABLE 3 Percentage of fluorescence intensities in Thioflavin-stainedpancreas sections of NORBA mice, vaccinated and unvaccinated. HighMedium Low Animal intensity % Intensity % intensity % Control Aβ⁻ mouse0 11.65 +/− 1.6  88.5 +/− 9.1 Aβ⁺, 18-month old 3.271- +/− 0.3 34.62 +/−3.5 62.11 +/− 6.1 unvaccinated mouse Aβ⁺, 18-month old,   0.74 +/− 0.0734.63 +/− 3.5 64.63 +/. 6.4  vaccinated mouse

Each value is the average of 5 countings. It appears that vaccinationreduces dramatically (by more than 70%) the high intensity fluorescencein the Thioflavine-stained pancreas sections of the NORBA transgenicmice with fully developed β-amyloid plaques. The medium and lowintensity fluorescence, which is unspecific since it is detected also inthe negative controls, remains unchanged upon vaccination, There is nohigh intensity fluorescence, in the negative controls.

In vitro experiments were conducted to investigate the mechanism bywhich the “autoantibodies” disintegrate the plaques. β-amyloidaggregates using the sequence Aβ (1-28) were made by incubating for 7days at 37° C. in phosphate buffered saline (PBS) at pH=7.1. This is thesequence used for the immunization of the mice. These deposits weredetectable by staining with Thioflavin T (“Th T”). Th T stainsamyloid-like deposits and exhibits enhanced fluorescence emission at 482nm and a new excitation peak at 450 nm when added to a suspension ofaggregated β-sheet preparations (Solomon et al. (1997) PNAS 94,4109-4112). FIG. 7 shows comparatively the disaggregation of β amyloidfibers by the 6C6 mAB and by sera from mice immunized with palmitoylatedAβ (1-16) reconstituted in liposomes.

It appears that both antibodies have solubilizing properties, but themonoclonal antibodies has a significantly stronger effect than the serumfrom the immunized mice, which contain polyclonal antibodies.

Any patents or other publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. One skilled in the art will readily appreciate thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned and inherent within. Thepresent examples along with the methods, procedures, treatments,compositions, and specific compounds described herein are presentlyrepresentative of preferred embodiments, and are not intended aslimitations on the scope of the invention. Those skilled in the art willknow or will be able to ascertain many equivalents to the specificembodiments of the invention described in the examples above using onlyroutine experimentation. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention as defined by the scope of the claims.

1. A tetra-palmitoylated beta-amyloid peptide having an N terminus and aC terminus.
 2. The tetra-palmitoylated beta-amyloid peptide of claim 1,wherein at least one of the palmitoyl moieties is on a lysine residueforming a palmitoylated lysine residue.
 3. The tetra-palmitoylatedbeta-amyloid peptide of claim 2, wherein the palmitoylated lysineresidue is at the C-terminus of the tetra-palmitoylated beta-amyloidpeptide.
 4. The tetra-palmitoylated beta-amyloid peptide of claim 3,having a second palmitoylated lysine residue at the C-terminus oftetra-palmitoylated the beta-amyloid peptide.
 5. The tetra-palmitoylatedbeta-amyloid peptide of claim 2, wherein the palmitoylated lysineresidue is at the N-terminus of the tetra-palmitoylated beta-amyloidpeptide.
 6. The tetra-palmitoylated beta-amyloid peptide of claim 4,further comprising a second palmitoylated lysine residue at theN-terminus of the beta-amyloid peptide.
 7. The tetra-palmitoylatedbeta-amyloid peptide of claim 2, wherein each of the four palmitoylmoieties are on lysine residue forming four palmitoylated lysineresidues such that the tetra-palmitoylated beta-amyloid peptide has apalmitoylated lysine residue is at the C terminus and a palmitoylatedlysine residue at the N-terminus.
 8. The tetra-palmitoylatedbeta-amyloid peptide of claim 7, having two palmitoylated lysineresidues at the C-terminus and two palmitoylated lysine residues at theN-terminus.
 9. The tetra-palmitoylated beta-amyloid peptide of any ofclaims 1-8, wherein the beta-amyloid portion of the tetra-palmitoylatedbeta-amyloid peptide is the beta-amyloid 1-16 peptide.
 10. A compositioncomprising the tetra-palmitoylated beta-amyloid peptide of any one ofclaims 1 through 8, wherein the tetra-palmitoylated beta amyloid peptideis anchored in a liposomal bilayer.
 11. The composition of claim 10wherein the liposomal bilayer is a liposome.
 12. A pharmaceuticalcomposition comprising the tetra-palmitoylated beta-amyloid peptide ofany of claims 1-8 and a carrier.
 13. A pharmaceutical compositioncomprising the composition of claim 10 and a carrier.
 14. A method fortreating an amyloid-associated disease in a subject, comprisingadministering to the subject an effective amount of thetetra-palmitoylated beta-amyloid peptide of any of claims 1-8.
 15. Themethod of claim 14, wherein the amyloid-associated disease isAlzheimer's disease.
 16. The method of claim 14, wherein theamyloid-associated disease comprises Type 2 diabetes mellitus, amyloid A(reactive), secondary amyloidosis, familial mediterranean fever,familial amyloid nephropathy with urticaria and deafness, Muckle-wellsSyndrome, myeloma, macroglobulinemia associated Muckle-Wells Syndrome,chronic hemodialysis, ATTR, familial amyloid polyneuropathy, familialamyloid cardiomyopathy, isolated cardiac amyloid, systemic senileamyloidosises, AIAPP, amylin insulinoma, atrial naturetic factor,procalcitonin, gelsolin, cystatin C, AApo-A-I, AApo-A-II,fibrinogen-associated amyloid, scrapie, Creutzfeld Jacob disease,Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis,or persons who are homozygous for the apolipoprotein E4 allele.
 17. Acomposition comprising the tetra-palmitoylated beta-amyloid peptide ofclaim 9, wherein the tetra-palmitoylated beta amyloid peptide isanchored in a liposomal bilayer.
 18. The composition of claim 17 whereinthe liposomal bilayer is a liposome.
 19. A pharmaceutical compositioncomprising the tetra-palmitoylated beta-amyloid peptide of claim 9 and acarrier.
 20. A pharmaceutical composition comprising the composition ofclaim 17 and a carrier.
 21. A method for treating an amyloid-associateddisease in a subject, comprising administering to the subject aneffective amount of the tetra-palmitoylated beta-amyloid peptide ofclaim
 9. 22. The method of claim 21, wherein the amyloid-associateddisease is Alzheimer's disease.
 23. The method of claim 21, wherein theamyloid-associated disease comprises Type 2 diabetes mellitus, amyloid A(reactive), secondary amyloidosis, familial mediterranean fever,familial amyloid nephropathy with urticaria and deafness, Muckle-wellsSyndrome, myeloma, macroglobulinemia associated Muckle-Wells Syndrome,chronic hemodialysis, ATTR, familial amyloid polyneuropathy, familialamyloid cardiomyopathy, isolated cardiac amyloid, systemic senileamyloidosises, AIAPP, amylin insulinoma, atrial naturetic factor,procalcitonin, gelsolin, cystatin C, AApo-A-I, AApo-A-II,fibrinogen-associated amyloid, scrapie, Creutzfeld Jacob disease,Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis,or persons who are homozygous for the apolipoprotein E4 allele.